BIOLOGY

OF

REPRODUCTION

14,405-411

Physiological

(1976)

Parameters KENNETH

of the Sea Urchin

W. GREGG2

and

Acrosome

CHARLES

Reaction’

B. METZ3

ABSTRACT The electron microscope was used in quantitatively assaying acrosome reactions of sea urchin sperm (Arbacia punctulata and other species) after exposure to solutions of egg jelly. Several physiological parameters which affected the precision of the assay were investigated. Each experiment was repeated at least three times and a minimum of 100 sperm were counted for each experimental variable. Replicate counts of sperm on the same or different grids were within 99 percent confidence limits of the original count. The percentages of acrosome reactions induced by solutions of egg jelly were consistently about 80 percent if fresh sperm were used and proper conditions were maintained. Percentages of acrosome reactions in sperm exposed only to sea water were rarely more than 10 percent. The time period during which acrosome reactions were induced coincided with the period of sperm agglutination induced by egg jelly solutions. Although egg jelly-induced sperm agglutination occurred well in the pH range 6.5-9.0, only low percentages of acrosome reactions occurred at pH levels below 7.5. At pH 8.5 and above, acrosome reactions occurred spontaneously in sea water. Consistently high percentages of acrosome reactions could not be obtained when sperm and egg jelly solutions were mixed unless the calcium content of the sea water was raised to 20-30 MM/mi. Sperm of species other than A. punctulata had different calcium requirements. The minimum concentration of egg jelly, measured in terms of g fucose/mi, necessary to induce maximum levels of acrosome reactions in A. punctulata was 0.5 Mg/mI.

glutinate and ther observed

INTRODUCTION

There logical

have

been

studies

(see tions

of

a large the

number

sperm

of morpho-

acrosome

Dan, 1970, for review), but to an understanding of the

the

reaction.

correctly that sperm tips had

Dan

(1952,

interpret discharge

1954)

several

species,

few contribuphysiology of was

Popa’s (1927) a small granule

when mixed with egg jelly observed the acrosome Dan

examined

reaction

the

first

to

observation from their

solutions. reaction. whole

agglutination. agglutination

were

separate

were

water,

Dan

the

reacted. found

acrosome

Accepted

Received

‘Contribution

In that

reaction,

calcium-deficient sperm

did

although

undergo

they

did

12, 1975. 14, 1975. No. 260 from

of

egg

Institute

results the

she

jelly

Haino Austin

solutions

relationship agglutination

between and the

reaction

events.

and (1962)

induced

sperm. experiments

concluded

acrosome

independent

in homologous quantitative

and Dan’s

Dan (1961) confirmed acrosome

The

experiwork. and that

reactions

aim of most was to determine

fertiizin-induced acrosome reaction.

of

the the

sperm How-

ever, before such experiments can be evaluated critically, the precision of the acrosome reaction assay must be established. Systematic quantitative examinations of the conditions

ag-

necessary the

and

Collier (1959), Piatigorsky and

December April

these and

She furinclud-

hyperalkalinity, without causing

Several quantitative observations ments have been reported since

sea

not

capacity. stimuli,

glass and reactions

From

that

fixed sperm in the electron microscope after exposure to sea water solutions of homologous egg jelly. She observed that many sperm acrosomes

fertilizing nonspecific

ing contact with induced acrosome

Popa Using

mounts

lose that

of

for

a precise

existing reports. responsiveness and

Molecular and Cellular Evolution. Support from the National Institute of General Medical Sciences (predoctoral fellowship 7-F1-GM-28, 960-02A1) and NSF grant GB 3899 is gratefully acknowledged. This work was submitted to the Graduate School, University of Miami, in partial fulfillment of the requirements for the Ph.D. degree. 2Present address: Dept. of Biology, Winthrop College, Rock Hill, South Carolina 29730. 3Present address: Institute of Molecular and Cellular Evolution, University of Miami, Coral Gables, Florida 33134.

were ably sulted assay. of

the

The clude:

often obtained. These reflect uncontrolled in less than optimal In

the

assay

work has

a)

the

are

lacking

here

the

quantitatively parameters

speed

of

the

in the

in sperm of reactions

variations variables conditions

reported been

physiological

responsiveness c) the effect 405

assay

Large variations low percentages

presumwhich refor the precision evaluated.

examined reaction;

inb)

the

of freshly shed and aged sperm; of pH during the reaction and

GREGG

406

upon

sperm

necessity e)

the

prior

of

calcium

concentration

maximum

levels

to

the

ions of

reaction;

for egg

d)

AND

METZ

the

the

reaction;

and

jelly

necessary

for

of reactions.

MATERIALS

AND

METHODS -

Collection

of

I-.:

Gametes

The sea urchins used in this work were collected in the area of Virginia Key, Florida. All were native to this area except Arbacia punctulata which had originally been obtained from Panama City, Florida. Spawning was induced in Arbacia punctulata by a ten volt (A.C.) electrical shock, and in the urchins, Tripneustes esculentus, Lytechinus variegatus, and Echino,netra lucunter, by injection of 3-5 ml of 0.5 M KCI into the body cavity. Eggs were shed into millipore-filtered sea water; sperm were collected with a Pasteur pipette and kept undiluted until use. Unless otherwise stated, results reported were obtained using A. punctulata.

Preparation

of

Egg Jelly

.5.

1

Solutions

Egg jelly solutions for use in the acrosome reaction assay were prepared in two ways. First, the supernatant sea water of unfertilized eggs was collected several hours after spawning and filtered through Whatman #1 filter paper. Second, the pH of freshly shed egg suspensions (usually 20 percent v/v) was lowered to 4.7-4.9 by the addition of 1 N HCI with constant stirring. After the eggs settled, the supernatant egg jelly solution was decanted, filtered through Whatman #1 filter paper and adjusted to pH 8.2 with 1 N NaOH. Both methods of preparation yielded jelly solutions equally capable of inducing the acrosome reaction. In most experiments reported here, jelly solutions used were obtained by the second procedure.

Acrosome

Reaction

Assay

Acrosome reaction assays were performed as described by previous workers (Dan, 1952, 1954, 1956; Collier, 1959; Haino and Dan, 1961; Piatigorsky and Austin, 1962). One drop of a 10 percent (v/v) sperm suspension was mixed with ten drops of a solution to be assayed. Within one minute following mixing, the sperm were rapidly mixed with sufficient 37 percent formaldehyde to bring the final concentration to 5-6 percent. Sperm suspensions were stored in fixative without damage for periods varying from one day to four months. Sperm were mounted on formvar-coated grids and allowed to dry. Excess sea water salts were removed with distilled water. Grids were examined in the Philips EMU 100-B electron microscope at approximately 7,000 X. Sperm were scored as unreacted and reacted (Plate 1). Those with complete or partial acrosomal filaments were classed as reacted. If the acrosomal region was obscured, the sperm was not counted.

PLATE Appearance of mount in electron X 51,300; (b)-reacted

I

sperm acrosomal region microscope. (a)-unreacted sperm, X39,900.

as whole sperm,

RESULTS Precision

of

the

Assay

The percentages counted in sequential sperm from the same

of

acrosome reactions observations of 100 sample were nearly always

SEA URCHIN

within the first count. sperm

99 percent confidence This precision was

sample

different

was

of the

Controlled

aging

formed

on

sperm undergo

showed the

hours ments, percent

at room however, suspension

on

the

were

sperm;

Reacted

or

Although the neous acrosome to

data

fall

had little

as

of ability to after several

were acrosome when

water

for

periods

Using this responsiveness

assay, of

occur

there sperm

tested

in (Fig.

sea 1).

here, spontasuspensions up

minutes never exceeded 10 percent. ly, Collier’s (1959) suggestion that reactions might been substantiated.

used at reac-

the

been diluted as 10 mm

are not shown reactions in sperm

sea

such

to

80

Accordingspontaneous in aged

ation

from

the error

mean was

was 2.3

10.7

percent

and

began

mixing

did

not

to appear sperm

increase

seconds). seconds

and

over

the

Agglutination and reversed

A few minutes water tended to and

to

fore,

efforts

reversal

after adhere

undergo

the

were of

two egg

to three jelly

time

span

began by about

dilution, to glass

acrosome

made

visible

solu-

tested

at

about 15 sec-

sperm or plastic

in sea tubes

to fix

reaction;

there-

immediately

agglutination.

after Generally,

sperm mixed with egg jelly solutions did not adhere to glass tubes as did sperm mixed with sea water. Likewise, sperm in calcium-deficient sea

water

did

adherence not occur

to determine were unsuccessful.

pH

not

adhere

of sperm consistently. the

to

to the For

glass

tubes.

The

sides of tubes did this reason, efforts

cause of the phenomenon The problem was minimized

fixing

sperm

within

the

solution

being

60 seconds

after

mixing

assayed.

Dependence

The ability some reaction

Over a period of several months, using three different jelly samples kept at 4#{176}C, more than 20 different males showed an average of 82.5 percent acrosome reactions. The standard devistandard

after

onds.

with

was little variability in from different males.

Reaction

Maximum percentages of acrosome reacwere attained within 15 seconds (Fig. 2). percentage of acrosome reactions in sea

(60 three

by

sperm

407

sperm

seconds

water

per-

In most experidiluted to a 10 after spawning.

the diluted suspensions the percentages of to

not

however,

temperature. sperm were immediately

suspensions even for

exposed

experiments

tended

of the

tions. tions The

no evident loss acrosome reaction

obtained

acrosome has not

same

REACTION

Speed

of the if the

Sperm

undiluted

If possible, once, since sperm water,

limits obtained

grids.

Responsiveness

tions

counted

ACROSOME

of in

sperm to undergo the the pH range 6.5-9.0

acrowas

the

percent.

S

T,.,,}

U 411,!

FIG.

1.

Effect

on

II

U

1

S

5!

acrosome

reactions

of

aging

three different sperm suspensions at room temperature (approximately 25#{176}C) in a 10 percent suspension. Sperm were diluted in sea water at time zero. The acrosome reaction assay was performed at time zero and at 10 mm intervals on each sperm suspension. One hundred sperm were counted for each point. Ninetyfive per cent confidence limits are indicated.

,II

,.q

FIG. 2. The percentage of acrosome reactions obtained at various times after mixing sperm and egg jelly solutions. Each curve represents a different egg jelly solution tested on the given date. Agglutination began at about three to five seconds after mixing and reversed at about 15 seconds after mixing. One hundred sperm were counted for each point. Ninetyfive per cent confidence limits are indicated. The percentage of reactions shown at time zero were those obtained in sea water alone. The percentage of acrosome reactions in sea water-treated sperm after 60 seconds (not shown) were not significantly higher than those at time zero.

GREGG

408

AND

METZ

N I 1 ‘I,

I

S

I,I_.

SI N

I

:

a II

II

IS

tSi

SW 11

II

4 U 11

FIG. 3. Effect of pH on specific and nonspecific induction of acrosome reactions. This is a summary of three experiments. Each point represents 600 sperm. Ninety-nine per cent confidence limits are indicated. The percentages of specifically induced reactions at each pH value were obtained by subtracting the per cent reactions in sea water from those in egg jelly at that pH.

assessed in three experiments pH 6.5 did not respond degree. The percentage of of sperm exposed to egg at pH 8.0, approximately water. tions sea

Significant were found water

(pH

percentages specifically were

percentages in sperm 8.5

and

reactions

percentages solutions.

of acrosome Fertiizin-induced occurred a single

reaction

inducing

Calcium

Dependence

A.

Consistent punctulata

calcium

was (12

Therefore, tulata, the tion

was

the sea

6.5

results were

the

Ca++

the

egg

jelly and

pH range 6.5 sperm exposed

at pH

and

that in the

adding

(Fig.

4 iWi.

1511

1511

IWII

‘Ulu

.1w1i

4

I

.15,1

4

I

FIG. 4. Percentages of acrosome reactions obtained sperm were diluted in sea water at pH 6.5 and subsequently exposed to egg jelly solutions at pH 6.5 or 8.0. The first four bars represent sperm which were suspended in unbuffered sea water at pH 6.5, for 45 seconds followed by the addition (indicated by arrows) of equal volumes of egg jelly or sea water (unbuffered at pH 6.5 or buffered at pH 8.0). The addition of buffered egg jelly or sea water to the unbuffered acidic suspensions resulted in a final pH of 7.8. The sperm were then fixed within 60 seconds. The last four bars represent control sperm. In these cases, the unbuffered and buffered solutions were mixed before adding the sperm. The sperm were then fixed and examined. One hundred sperm were counted for each treatment. Ninety-five per cent confidence limits are indicated. when

4).

concentration

by 9

to approximately

The

relationship and percentages

tion induced

by

ages

of

12

jelly

prepara-

5. At least 30 JAM of to obtain high percent-

reactions.

Additions

of

calci-

trations. acrosomes

In preliminary of E. lucunter

experiments, sperm reacted maximally in

solutions approximately

of

tions

from

egg

concentrareactions

experiment) caused a Only sperm of A. high calcium concen-

solu-

calcium

different

in Fig. necessary

acrosome

test

Dan (1954), percent. This

calcium acrosome

um above 48 JAM/mi (one small decrease in reactivity. punctulata required such

(approximately

sea area).

46 j.tM/ml. between of

three

tions is shown Ca4 +/ml were

with the

with A. puncCa++ concentraisotomic

JAM/mi

assay unless

of normal collecting

experiments sea water

to to

found acrosome

7.8

other

chloride (0.37 M) as suggested to a final concentration of increased

of

from

in the acrosome not obtained

by

pH,

induced that pH

percentages

were subsequently maximally to the

raised above JAM CaImi

increased

at

water

the

of egg jelly

in most normal

a given

reactions in agglutination

substance

content

tions water

For

reactions solutions

well in experiment,

sea water at pH capable of reacting

1511

4 1511

of acrosome reacexposed to alkaline 9.0).

in

at

solutions peaked pH of normal sea

by subtracting

acrosome

reversal 9.0. In

jelly the

of acrosome by egg jelly

obtained

(Fig. 3). Sperm to any significant acrosome reactions

Jil

did

not

homologous egg 12 JAM Ca/ml. 46

increase

(Fig.

Sometimes

70-80 underwent in Moore’s

Ca/ml)

concentration)

of

acrosome

reac-

of calcium on sperm acrosomes and T. esculentus was determined.

variegatus reactions

JAM

final

percentages

6).

The effect L. variegatus

(Cavanaugh, artificial

JAM/mi

jelly containing Excess calcium

and

1956) natural nearly

percent

of

sperm

spontaneous calcium-free containing sea water 100

percent

of

of L.

acrosome sea water no egg jelly. (containing of

the

sperm

In 12

SEA

URCHIN

ACROSOME

REACTION

409

II I. #{149}6

70 I-h-’

I0 Ractioss

40 30 C.k

1.h-,.:..

20

.11.1)

FIG. 5. Relationship between calcium concentration and the percentage of Arbacia punctulata sperm acrosome reactions induced by homologous egg jelly. The calcium concentration of Moore’s calcium-free sea water was adjusted by the addition of 0.37 M CaCI2. The addition of 10 percent egg jelly solution (v/v) (previously diluted 1:6 in calcium-free sea water) to the test solution resulted in an insignificant increase in the calcium concentration (approximately 0.2 MM/mI). Two hundred sperm were counted for each point and ninety-nine per cent confidence limits are indicated.

The percentages of reacted acrosomes of sperm treated with sea water at the various calcium concentrations (not shown) were never significantly higher than those treated with calcium-free sea water.

L. known

variegatus whether

of

um levels of fixation.

is a natural Sperm

played

greater

sperm

of

tions

was 6

sensitivity

Concentration

(PCyRI) by

E.

sperm

A.

egg

Effect

of

low

(12

MM/mI)

is

and

high

(48

percentage acrosome reactions of sperm lucunter. This is a summary of four experiments. Each bar represents a total count of 800 sperm. Ninety-nine per cent confidence limits are indicated. MM/mI)

calcium

concentrations

the of Echinometra on

identical

(approximately

for EJb). third egg

However, in preliminary results with jelly solution, the acrosome reaction

inducing titer was

titer was 8,000.

128

1,000

while

for

the

Eja

and

64 a

agglutinating

jelly

A solu-

of T. esculentus

sea

water

containing

punctulata

was the

of Dische

determined

S

huts..

dilutions

acrosome of

method Figure

0.5 JAg fucose/mI or more. preparations, the titers activity

and

8 shows

reactions

these

egg

of

prefrom after

colorimetric

et al. (1949). of

pentose, solutions

jelly

Maximum percentages of acrosome were induced by egg jelly solutions

inducing

6.

SW ish

did

higher than 6 JAM/mI, of nonspecifically induced were observed (Fig. 7).

using

percentage serial

than lucunter.

to

concentration of methyl fucose, in two egg jelly of

FIG.

SW IosCi

of Egg Jelly

hydrolysis the

calcium

or

In

calcium concentrations high percentages acrosome reactions

eggs

to

with

Ca/mi.

is not calci-

11’ EJ Iowti

occurrence or an artifact T. esculentus also dis-

of

obtained

JAM

The sumably

reacted. It to normal

A. punctulata acrosome response

maximal

at

consistently this sensitivity

II

induced solutions. reactions containing

In both of these jelly acrosome reaction

agglutinating

activity

were

1.5,.

1,ut.

(.1/.4)

FIG. 7. Relationship between calcium concentration and the percentage acrosome reactions obtained upon exposure of sperm from Tripneustes esculentus to homologous egg jelly solutions. This is a summary of three experiments, using three different animals (A, B, and C). The sea water solutions containing various amounts of calcium were made by fortifying Moore’s calcium-free sea water with appropriate volumes of 0.37 M CaCI2. Each point represents a total count of 600 sperm. Ninety-nine per cent confidence limits are indicated.

GREGG

410

AND

METZ

coat

the

them.

glass

Such

or

free-swimming sampling errors.

S

some water

plastic

coating

reactions ranged

sperm When of from

IUiEi

FIG.

8. Relationship

CIKUUaI

(M/S)

between fucose reactions for

and

concentration two egg jelly

percentage acrosome samples. Fucose was determined after aliquots of the two egg jelly samples, using the colorimetric method of Dische The decline of acrosome reaction inducing agglutinin activity was similar. Each point total count of 100 sperm. Ninety-five confidence limits are indicated.

hydrolysis

of

Eja and EJb et al. (1949). activity and represents a per cent

unit on significant

either side decreases

tions

The tion

quantitative assay

several reliable. tus

as

sea used

urchin Collier

urchin

by

previous

species (1959),

purpuratus,

not

that

from

following

24-60 exposure

no data

on

acrosome

to egg

control

sperm

jelly.

with

acrosome large

suspensions.

Haino

ty in sperm physiological Collier sperm,

reactions variations

suggested uncontrolled (e.g., pH, contact

factors

undoubtedly assay. effect

tative was

and

Dan

responsiveness maturity

tors sperm the the

between between

of

reactions

They

exposed

alone. Piatigorsky and Austin punctulata, similarly reported of

of exposed 3-21. ranged

Haino and Dan depressus and reported varia-

percent

to sea water

of in

pH levels

8.0 of

resulted specifically

solutions

were

not

due

in

in perreac-

to irreversi-

a clearly

defined

acrosomal

filament,

control

of

calcium

sperm of esculentus

to

concentration

A. punctulata, E. were not induced

was

use of reactions

the in

lucunter and by homologous

T.

egg jelly calcium

solutions unless threshold were present. If these

exceeded punctulata

in sperm and egg jelly mixtures and E. lucunter there was

crease

in

sea

percentages

of

low percentages induced in the water

and

high

have

levels of levels were

acrosome

of A. no in-

reactions.

of control

acrosome sperm

levels

of

reactions exposed to

calcium.

If

with in

sperm of sea water

the

with A. levels

calcium threshold was exceeded T. esculentus, the control sperm

50-60 different

percent, sperm

began to undergo acrosome reactions. Sperm of L. variegatus were often, but not consistently, found to have high percentages of reacted

attributed to the

variabili-

differences sperm,

the

reported here these variables

of

in while

acrosomes after water. Experiments

in calcium-free L. variegatus were

sea not

pursued The

for this reason. high calcium requirement

and these

reactions present seasonal

with sperm of A. punctulata was not early in the breeding season. Therefore, fluctuations in calcium requirements

precision

of

elucidates in quanti-

may

exist,

in sea water tended to

but

investigated. sperm

sperm sperm

dilution with

the fac-

terms.

Speed of handling diluted important because the

obtain

(1962), maximum

calcium concentration) with surfaces. All

The work of some of

of

Only were

reported

a lack of speed in handling chemical and physical

influenced

to

levels of specifically induced Variations of one-half pH

extremely important for successful assay. Maximum levels of acrosome

percentages

in control sperm ranged from about egg jelly solutions

only to sea This prob-

the entire acrosomal region appeared ruptured and no filaments were observed. The

highly

of led to acro-

acrosome reactions. Decreases of specifically induced acrosome in acid

hold

ble sperm damage. In very basic solutions (pH 8.5 and above) the nonspecifically induced acrosome reactions were morphologically different from specifically induced reactions. In-

with

been

Strongylocentro-

from about 30-60 percent. (1961), using Pseudocentrotus Hemicentro tus pulcherrimus, tions

reac-

workers

has using

reported

acrosome reactions only to sea water Those exposed to

acrosome

exposed percent.

10 percent or less. of pH was necessary

high reactions.

induced centages

to

numbers

by fixing sperm one-half to mixing with the assay solucontrol levels of acrosome

consistently acrosome

stead

ON

DISCUSSI

of

used the

and may have coating occurred,

sperm 10-20

lem was minimized two minutes after tions. This assured reactions Control

tubes

depleted

and to

The

acrosome jelly

were

relative reactions

P. depressus egg

they

(early as

reported

for

not

acrosome

systematically

insensitivity of in the by

H.

of

the

pulcherrimus

breeding Haino

season) and

Dan

SEA

well

have

(1961)

may

calcium of the

requirements sperm to

obvious per that

been

to

ACROSOME

reported assay for

is

and for

experiments

relating

acrosome

reaction

jelly solutions provide clear-cut

to

ever, of

that

at

acrosome

0.5

cose mum

agglutinating activities

of egg

fucose content It was apparent,

jig fucose/mI, were

Piatigorsky 200 times

did

maximum induced. and this

not howlevels

Using

Austin amount

the

(1962) of fu-

was necessary in order to induce maxilevels of acrosome reactions. The ap-

parent conflict be explained Austin

of by

used

been ethanol not

the results.

reactions

same species, reported that

the

inducing

egg

these results the fact that jelly

concentrated and dialyzed. determine

procedures

samples

by

acrosome reaction jelly samples.

any

which

had

precipitation These workers

whether had

can probably Piatgorsky and

such

concentration

deleterious

inducing

with did

activities

effect

on of

egg

M.

ed. Formulae Laboratory

fertilizin

on

puratus. 163-170.

Acta

the

sperm

and Methods IV of tbe (Woods Hole, Mass.;

The effect of homologous of Strongylocentrotus pur-

Embryol.

et.

Morph.

Exper.

2.

J. C. (1952). Studies on the acrosome. I. Reaction to egg s ter and other stimuli. Biol. Bull. 103, 54-56. Dan, J. C. (1954). Studies on the acrosome. Ill. Effect of calcium deficiency. Biol. Bull. 107, 335-3 39. Dan, J. C. (1956). The acrosome reaction. Int. Rev. Cytol. 5, 365-393. Dan, J. C. (1970). Morphogenetic aspects of acrosome formation and reaction. In: Advances in Morphogenesis, Vol. 8. Eds. M. Abercrombie, Jean Brachet, and Thomas J. King. (Academic Press, New York). pp. 1-40. Dische, Z.. Shettles, L. B. and Osnos. M. (1949). New specific color reactions of hexoses and spectrophotometric micromethods for their determination. Arch. Bioch. Biophys. 22, 169-184. 1-laino, K. and Dan, J. C. (1961). Some quantitative aspects of the acrosomal reaction to jelly substance in the sea urchin. Embryologia 5, 376-383. Piatigorsky, J. and Austin, C. R. (1962). Relationship of fertilizin to acrosome reaction in Arbacia. Biol. Bull. 123,473. Pops, G. T. (1927). The distribution of substances in the spermatozoon (Arbacia and Nereis). Biol. Bull. 52, 238-257. Dan,

The

G.

Marine Biological 1956). Collier, J. R. (1959).

conthat

species.

and

411 REFERENCES

Cavanaugh,

in this paany species

depends upon knowledge the calcium requirements

REACTION

varying

than inability reaction. It

the

from the results success of the

of urchin trol of

due

rather undergo

URCHIN